Cell Navigator® Lysosome Staining Kit *Green Fluorescence*

Protocol summary

1. Prepare cells
2. Add LysoBrite™ Green working solution
3. Incubate at 37°C for 30 minutes to 2 hours
4. Analyze the cells under fluorescence microscope at Ex/Em = 490/525 nm (FITC filter set)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

Add 20 µL of 500X LysoBrite™ Green stock solution (Component A) to 10 mL of Live Cell Staining Buffer (Component B) to make LysoBrite™ Green working solution. Protect from light. Note: 20 µL of 500X LysoBrite™ Green (Component A) is enough for one 96-well plate. The optimal concentration of the fluorescent lysosome indicator varies depending on the specific application. The staining conditions may be modified according to the particular cell type and the permeability of the cells or tissues to the probe.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

For adherent cells:

1. Grow cells either in a 96-well black wall/clear bottom plate (100 µL/well/96-well plate) or on cover-slips inside a petri dish filled with the appropriate culture medium. When cells reach the desired confluence, add equal volume of LysoBrite™ Green working solution.
   
   
2. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes to 2 hours.
   
   
3. Observe the cells using a fluorescence microscope with FITC filter set (Ex/Em = 490/525 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained.

For suspension cells:

1. Centrifuge the cells at 1000 rpm for 5 minutes to obtain a cell pellet and aspirate the supernatant.
   
   
2. Resuspend the cell pellet gently in pre-warmed growth medium, and then add equal volume of LysoBrite™ Green working solution.
   
   
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes to 2 hours.
   
   
4. Observe the cells using a fluorescence microscope with FITC filter set (Ex/Em = 490/525 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained. Suspension cells may be attached to cover-slips that have been treated with BD Cell-Tak^® (BD Biosciences) and stained as adherent cells.